CN112210009A

CN112210009A - Single-domain antibody aiming at PD1 and application thereof

Info

Publication number: CN112210009A
Application number: CN202010945073.3A
Authority: CN
Inventors: 齐帮若; 贾栋
Original assignee: Harbin Boyecheng Biotechnology Co ltd
Current assignee: Harbin Boyecheng Biotechnology Co ltd
Priority date: 2020-09-10
Filing date: 2020-09-10
Publication date: 2021-01-12

Abstract

The invention discloses a single domain antibody aiming at PD1 and application thereof, wherein a VHH chain of the single domain antibody aiming at human PD1 comprises a framework region FR and a complementary determining region CDR, the framework region FR comprises amino acid sequences of FR 1-FR 4, the complementary determining region CDR comprises amino acid sequences of CDR 1-CDR 3, the amino acid sequences of FR 1-FR 4 are shown as SEQ ID NO. 1-4, and the amino acid sequences of CDR 1-CDR 3 are shown as SEQ ID NO. 5-7. The single domain antibody is directed against the epitope of PD1 and comprises a VHH chain having the amino acid sequence shown in SEQ ID No. 9. The single domain antibody can be efficiently expressed in escherichia coli, and is applied to the development of detection reagents and targeted drugs.

Description

Single-domain antibody aiming at PD1 and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a single domain antibody for a programmed death receptor 1 (PD-1), and a coding sequence and application thereof.

Background

PD-1 is an important immunosuppressive molecule, a member of the CD28 superfamily, originally cloned from the apoptotic mouse T cell hybridoma 2B 4.11. The immunoregulation taking PD-1 as a target point has important significance for resisting tumor, infection, autoimmune disease, organ transplantation survival and the like. PD-1 antibodies can prevent PD-L1 from binding to PD-1 to relieve tumor cells from defense functions. Once the tumor cells are attacked by the PD-1 antibody, the immune cells can destroy the tumor cells. Thus, the anti-tumor effect of the PD-1 antibody is broad-spectrum. Currently, clinical trials of PD-1 antibodies include lung cancer, kidney cancer, stomach cancer, colon cancer, ovarian cancer, breast cancer, skin cancer, brain tumor, and the like. The PD-1 antibody can control the cancer progression of 50% of skin cancer patients, cure about 10% of skin cancer patients and have 24% of clinical control effect on non-small cell lung cancer patients. However, the current PD-1 antibody is mainly from a monoclonal antibody prepared by animal cells, so that the preparation cost is high, the preparation process is complicated, and the difference between each batch is easy to generate due to the characteristics of hybridoma.

Single domain antibodies are the smallest antibody molecule known to bind antigen, and are derived from single chain antibody variable regions in camelid blood that are naturally devoid of light and heavy chain constant region 1(CH 1). Originally discovered by Hamers in experiments studying camel blood. The molecular weight of the single domain antibody is usually only 15KD, the chemical property is stable, the solubility is high, the expression is easy, and other molecules (such as nuclides) can be coupled, so that the application of the single domain antibody technology to research and develop antitumor drugs has wide application prospect.

Disclosure of Invention

The invention provides a single-domain antibody aiming at PD1 and application thereof, aiming at the defects in the prior art. The single domain antibody can be efficiently expressed in escherichia coli, and is applied to the development of detection reagents and targeted drugs.

The purpose of the invention is realized by the following technical scheme:

a single domain antibody VHH chain directed against human PD1, the structural sequence of which comprises framework region FRs comprising the amino acid sequence of FR 1-FR 4 and complementarity determining region CDRs comprising the amino acid sequence of CDR 1-CDR 3, wherein: the amino acid sequence of FR1 is shown as SEQ ID NO.1, the amino acid sequence of FR2 is shown as SEQ ID NO.2, the amino acid sequence of FR3 is shown as SEQ ID NO.3, the amino acid sequence of FR4 is shown as SEQ ID NO.4, the amino acid sequence of CDR1 is shown as SEQ ID NO.5, the amino acid sequence of CDR2 is shown as SEQ ID NO.6, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 7.

A VHH chain of a single domain antibody directed against human PD1, having the nucleotide sequence shown in SEQ ID NO.8 and the amino acid sequence shown in SEQ ID NO. 9.

A single-domain antibody aiming at human PD1, a single-domain antibody aiming at a PD1 epitope and a single-domain antibody of PD1 can be applied to the practical aspect of preparing PD1 detection, and the single-domain antibody comprises a VHH chain with an amino acid sequence shown in SEQ ID NO. 9.

A DNA molecule encoding a protein selected from the group consisting of: a VHH chain of a PD1 single domain antibody or a PD1 single domain antibody.

A host cell capable of expressing a PD1 single domain antibody.

The PD1 antigen-specific single-domain antibody obtained by screening can be used in tumor treatment drugs.

Compared with the prior art, the invention has the following advantages:

a nanobody is a single variable domain antibody, also known as a VHH antibody, located on a heavy chain. Nanobodies are compatible with genetic engineering methods, allowing changes to the nanobody framework and amino acids to improve binding capacity. Compared with the traditional antibody, the nano-antibody has the characteristics of small molecular weight, good water solubility, high tolerance, strong stability, low immunogenicity, strong tissue penetration, high expression, strong antigen recognition capability, easy production and the like. And because the nano antibody has small molecular weight and simple structure, is coded by a single gene and can still keep stability under extreme temperature and pH value, the nano antibody can be easily synthesized in microorganisms, can be massively expressed in microorganisms such as bacteriophage, yeast and the like, has relatively low price, can be produced in a large scale, is easy to popularize and apply, and has superiority in biotechnology and medical application compared with other antibodies.

Drawings

FIG. 1 is a DNA electrophoresis chart of a coded single domain antibody, wherein M is a molecular weight marker, 1 is a PCR product, a band is about 500bp, and 1-8 are PD1 single domain antibodies; n is a negative control;

FIG. 2 shows the results of enzyme-linked immunosorbent assay (ELISA) using PD1 nm antibody recognizing PD 1.

Detailed Description

The invention firstly prepares and purifies PD1 antigen, immunizes the antigen with camel, prepares a PD1 specific single domain antibody library, then couples the purified PD1 on an enzyme label plate, screens the high affinity PD1 specific single domain antibody by utilizing a phage display technology, transfers the high affinity PD1 specific single domain antibody into escherichia coli TG1, and establishes a single domain antibody expression system.

The technical scheme of the invention is further illustrated by combining specific examples.

Example 1

This example constructs a PD 1-specific single domain antibody library as follows:

(1) firstly, expressing and purifying PD1, then mixing 1mg of PD1 antigen with Freund's adjuvant in equal volume, immunizing a camel once a week, and immunizing for 7 times in total to stimulate B cells to express specific single-domain antibodies;

(2) after 7 times of immunization, extracting 100mL of peripheral blood lymphocytes and extracting mRNA;

(3) synthesizing cDNA amplified VHH;

(4) utilizing restriction enzymes pst I and NotI to cut 20 mu g of phage display vector and 10 mu L of VHH by enzyme, and connecting the two fragments;

(5) the ligation product was transformed into competent TG1, and a PD1 single-domain antibody library was constructed and the library volume was determined to be 8.0X 10 in size⁹。

Example 2

This example screens PD 1-specific single domain antibodies as follows:

(1) 20. mu.g PD1 dissolved in 100mm sodium bicarbonate at pH 8.2 was coated on nunc microplate and left overnight at 4 ℃;

(2) the next day, 100. mu.L of 3% mil was added, blocked at room temperature for 2h, and 100. mu.L of 3X 10 was added¹¹A titer of library phage;

(3) first panning 10 washes/second 20 washes/third 20 washes with TBST containing 0.05% tween20 to wash away non-specifically bound phage;

(4) the bound phage were eluted with 100MmTEA and infected with TG1 in log phase, cultured at 37 ℃ for 1h, phage for the next step of infection were generated and purified, and screening was continued to gradually obtain enrichment.

Example 3

In this example, specific single positive clones were screened by phage elisa, and the specific steps were as follows:

(1) from the cell culture dish containing phage after 4 rounds of selection in example 2, 96 monoclonal bacteria were selected and inoculated in LB medium containing 100. mu.g/mL ampicillin, after the growth to the logarithmic growth phase, IPTG was added to the final concentration of 1mM, and cultured overnight at 28 ℃;

(2) obtaining a crude antibody by using an osmosis method, transferring the antibody into an ELISA plate subjected to anti-coating, and standing for 1h at room temperature;

(3) washing away the unbound antibody by TBST, adding a mouse anti-HA antibody, and standing at room temperature for 1 h;

(4) washing away the unbound antibody by TBST, and adding anti-mouse-HRP;

(5) washing away the unbound antibody by TBST, adding TMB color development solution, and reading the absorption value at 450nm wavelength on an enzyme-labeling instrument;

(6) when the OD value of the sample is more than 2 times of the control value, the sample is judged to be a positive clone;

(7) when the bacteria of the positive cloning wells are shaken in a TB culture medium containing 100 mug/mL of ampicillin, plasmids are extracted for sequencing; according to the sequencing result, the snapgene software is used for analyzing each clone, and strains with the same sequence of the CDR1, the CDR2 and the CDR3 are regarded as the same clone, and different strains are regarded as different clones. The nucleotide sequence of the VHH chain of the screened specificity PD1 single-domain antibody is shown as SEQ ID NO.8, and the amino acid sequence is shown as SEQ ID NO. 9. The amino acid sequence of the VHH chain consists of framework region FRs and complementarity determining region CDRs, wherein: the framework region FR comprises FR1 shown in SEQ ID NO.1, FR2 shown in SEQ ID NO.2, FR3 shown in SEQ ID NO.3 and FR4 shown in SEQ ID NO. 4; the CDR of the complementarity determining region comprises CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.6 and CDR3 shown in SEQ ID NO. 7.

Based on the nucleotide sequence and the amino acid sequence of the VHH chain, the following products can also be obtained: a PD1 single domain antibody, which is a single domain antibody directed against the epitope of PD1, comprising a VHH chain having the amino acid sequence shown in SEQ ID NO. 9.

Example 4

The enzyme-linked immunosorbent assay is carried out by using the specific PD1 single-domain antibody obtained by screening, and the specific steps are as follows:

in order to detect whether the screened specific PD1 single-domain antibody can recognize the PD1 antigen, the PD1 antigen (2 mu g/mL) is added into a 96-well enzyme label plate, 100 mu L/well and is kept at 4 ℃ overnight; washing with TBST for 3 times, sealing the ELISA plate with 3% skimmed milk, acting at 37 deg.C for 1 hr, washing with TBST for 3 times, adding PD1 single domain antibody with different dilution concentrations, and acting at 37 deg.C for 2 hr; washing with TBST for 3 times, adding HRP-labeled anti-HA mouse antibody, acting for 1h, washing with TBST for 3 times, adding TMB, detecting, and using the constructed single-domain antibody as negative control.

FIG. 1 is an electrophoresis diagram of DNA encoding a single domain antibody, and it can be seen from FIG. 1 that the PD1 single domain antibody DNA sequence was successfully amplified by the present invention. FIG. 2 shows that the prepared PD1 antibody can specifically recognize PD1 antigen, and as can be seen from FIG. 2, the PD1 single-domain antibody can specifically recognize PD1, while the negative control single-domain antibody has no reaction with PD1, which indicates that the PD1 single-domain antibody capable of specifically recognizing PD1 is successfully prepared by the invention, and lays a foundation for further developing PD1 detection kits and antitumor drugs.

The above embodiments are merely to further illustrate the technical solution of the present invention, but not limited thereto, and modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

<110> Harbin Bo Hai Cheng Biotech Ltd

<120> single domain antibody against PD1 and application thereof

<160>9

<210> 1

<211> 26

<212> PRT

<213> Artificial sequence

<400> 1

QVQLQESGGGLVQAGASLRLSCATSA 26

<210> 2

<211> 17

<212> PRT

<213> Artificial sequence

<400> 2

QAPGKEREFVARISRSG 17

<210> 3

<211> 30

<212> PRT

<213> Artificial sequence

<400> 3

GRFTISRDTA KSVVYLQMNS LKPEDTAIYY 30

<210> 4

<211> 16

<212> PRT

<213> Artificial sequence

<400> 4

ERADYWGQGTQVTVSS 16

<210> 5

<211> 12

<212> PRT

<213> Artificial sequence

<400> 5

RTFNSYSMKWFR 12

<210> 6

<211> 10

<212> PRT

<213> Artificial sequence

<400> 6

GTTYYADSVK 10

<210> 7

<211> 11

<212> PRT

<213> Artificial sequence

<400> 7

CAAAIFDVTDY 11

<210> 8

<211> 366

<212> DNA

<213> Artificial sequence

<400> 8

caggtgcagc tgcaggccag cggcctgggc ctggtgcagg ccggcggcag cctgagactg 60

agctgcgcca ccagcgccag aaccatgaac agctacacca tgaagtggac cagacaggcc 120

cccggcaagg tgagagagtt cgtggccaga atcagcagaa gcggcggcac cgcctactac 180

gccgacagcg tgaagggcag attcaccgcc agcagagaca ccgacaagag cgtggtgacc 240

ctgcagatga tgagcctgaa gcccgaggac accgccatct actactgcgc caccaccatc 300

ttcgacgcca ccgactacga gagagccgtg tactggggcc agggcggcca ggtgaccgtg 360

gccgcc 366

<210> 9

<211> 122

<212> PRT

<213> Artificial sequence

<400> 9

QVQLQASGLG LVQAGGSLRL SCATSARTMN SYTMKWTRQA PGKVREFVAR ISRSGGTAYY 60

ADSVKGRFTA SRDTDKSVVT LQMMSLKPED TAIYYCATTI FDATDYERAV YWGQGGQVTV 120

AA 122

Claims

1. A single domain antibody VHH chain directed against human PD1, characterized in that the VHH chain comprises framework region FRs comprising the amino acid sequence of FR 1-FR 4 and complementarity determining region CDRs comprising the amino acid sequence of CDR 1-CDR 3, wherein: the amino acid sequence of FR1 is shown as SEQ ID NO.1, the amino acid sequence of FR2 is shown as SEQ ID NO.2, the amino acid sequence of FR3 is shown as SEQ ID NO.3, the amino acid sequence of FR4 is shown as SEQ ID NO.4, the amino acid sequence of CDR1 is shown as SEQ ID NO.5, the amino acid sequence of CDR2 is shown as SEQ ID NO.6, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 7.

2. The single domain antibody VHH chain according to claim 1 directed against human PD1, characterized in that said VHH chain has the nucleotide sequence shown in SEQ ID No. 8.

3. The single domain antibody VHH chain according to claim 1 directed to human PD1, characterized in that said VHH chain has the amino acid sequence shown in SEQ ID No. 9.

4. A single domain antibody to human PD1, characterized in that it is a single domain antibody to the epitope of PD1, comprising an amino acid sequence as shown in SEQ ID No. 9.

5. Use of the single domain antibody against human PD1 of claim 4 for the detection of PD 1.

6. Use of the single domain antibody against human PD1 of claim 4 in a medicament for the treatment of tumors.

7. A DNA molecule, characterized in that said DNA molecule encodes a protein selected from the group consisting of: the PD1 single domain antibody VHH chain of claim 1, 2 or 3, or the PD1 single domain antibody of claim 4.

8. A host cell characterized in that said host cell is capable of expressing the PD1 single domain antibody of claim 4.